Ionic liquids with a theophyllinate anion

Article abstract of DOI:10.1039/c4nj00463a

Ammonium and piperidinium theophyllinate-based ILs were synthesized and characterized. Physicochemical properties, such as thermal stability, phase transition temperatures, viscosity, density, refractive index, as well as surface activity, feeding deterrence, antifungal activity and also biodegradability were determined. The synthesized theophyllinate-based ILs were surface active compounds. They exhibited insect-feeding deterrent activities. At the same time, the studied salts are also efficient fungicides and potential pesticidal ionic liquids. The obtained data suggest that certain structural modifications may increase their biodegradability in order to provide an environmentally friendly product. the Partner Organisations 2014.

Full text of DOI:10.1039/c4nj00463a

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Ionic liquids with a theophyllinate anion†
Bartosz Markiewicz,a Agata Sznajdrowska,a Łukasz Chrzanowski,a
Cite this: New J. Chem., 2014,
38, 3146
Łukasz Ławniczak,a Agnieszka Zgoła-Grzeskowiak,a Krzysztof Kubiak,b Jan Nawrotb
´
and Juliusz Pernak*a
Ammonium and piperidinium theophyllinate-based ILs were synthesized and characterized. Physicochemical
properties, such as thermal stability, phase transition temperatures, viscosity, density, refractive index, as well
as surface activity, feeding deterrence, antifungal activity and also biodegradability were determined. The
synthesized theophyllinate-based ILs were surface active compounds. They exhibited insect-feeding deterrent
activities. At the same time, the studied salts are also efficient fungicides and potential pesticidal ionic liquids.
The obtained data suggest that certain structural modifications may increase their biodegradability in order to
provide an environmentally friendly product.
Received (in Porto Alegre, Brazil)
26th March 2014,
Accepted 15th April 2014
DOI: 10.1039/c4nj00463a
www.rsc.org/njc
xanthine family. This compound bears structural and pharmaco-
logical similarity to caffeine and is used in the therapy of
Introduction
Ionic liquids (ILs, defined as organic salts with a melting point respiratory diseases. Theophylline is naturally found in cocoa
below 100 1C) have been investigated for an increasing number beans and trace amounts are present in brewed tea. The high
of applications due to their unique properties.1,2 The evaluation concentration of caffeine in young tea leaves protects the plant
of ILs has proceeded very quickly from the first generation (with from virulent microbes16 as well as insects,17 therefore theophyl-
unique, designable physical properties), through the second line was selected as an anion with promising properties.
generation (with targeted chemical properties, combined with
ILs obtained from natural compounds have recently attracted
selected physical properties), to the third generation (with targeted increasing attention. The possibility to use anions originating from
biological properties, combined with physical and chemical natural or renewable materials to prepare ILs has been marginally
properties).3 ILs with biologically active ions were synthesized investigated. Natural amino acids have been used to develop ILs,
and shown to retain the original biological activity of both in which the anion is a natural compound.18 Additionally, ILs with
the cation and anion. They can be directly prepared from lactate19,20 and mandelate21 as anions have also been prepared.
pharmaceutically-active ingredients4–7 and also from commonly Herein, we present novel active ILs with theophyllinate as an
used pesticides.8–14 The regulations of the European Parliament anion. Ammonium cations with sp3 hybridisation and a long alkyl
state that low-risk plant protection products should be identified substituent were selected as counter ions based on the assumption
and placed on the market.15 Regulatory pressure to eliminate the that hydrophobic, non-crystalline and biologically active salts will
most toxic, older synthetic pesticides in order to reduce the be obtained. The aim of the study was to search for efficient and
environmental impact is helping to drive the development of non-hazardous pesticidal ionic liquids.
pesticides based on natural products.
Recently, herbicidal ionic liquids seem to be an interesting
proposal.8,10–14 They allow for reducing the herbicide dose per
hectare, while controlling its toxicity (toxic phenoxy-based
Results and discussion
herbicides may become nontoxic as herbicidal ionic liquids).8 Ammonium (1–12) with various substituents and piperidinium
Theophylline (3,7-dihydro-1,3-dimethyl-1H-purine-2,6-dione (13–14) theophyllinates were synthesized with a high yield of
also known as 1,3-dimethylxanthine) is a member of the 92–98% (Table 1). The structures of the 14 synthesized theophyl-
linate salts are shown in Fig. 1. The majority of the tested salts
are fundamentally novel, as only a single compound (9) has been
a Faculty of Chemical Technology, Poznan University of Technology,
tested in previous studies.22 All salts are waxes or liquids at room
M. Skłodowskiej-Curie 2, 60-965 Poznan, Poland.
temperature, except 1, which is a solid with a melting point
E-mail: juliusz.pernak@put.poznan.pl; Fax: +48 61 665 3649
above 100 1C (116 1C, Table 2). The above-mentioned liquids and
waxes might, in some cases, be considered to represent ILs. The
synthesized ILs were stable in air and in contact with water and
b Institute of Plant Protection, National Research Institute, Poznan 60-318, Poland
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c4nj00463a
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Table 1 Prepared theophyllinates
the water content was determined to be less than 500 ppm
by colorimetric Karl-Fischer titration. The obtained ILs were
insoluble in hexane and ethyl acetate but were soluble in
acetone, dichloromethane, DMSO and low molecular weight
alcohols. All of the synthesized salts, with the exception of salts
7 and 8, were exquisitely soluble in water. The purity of ILs with
a long alkyl substituent was determined by a direct two-phase
titration technique EN ISO 2871-2: 2010 and expressed as the
content of the cationic active substance (Table 1). The purity of
the obtained ILs was high (497%). All of the prepared ILs were
characterized by 1H NMR and 13C NMR and elemental analysis.
The chemical shift of the proton between the two nitrogen
atoms in the imidazole ring reached lower values and covered a
range of 0.65–0.84 ppm. The proton chemical shift value for
theophylline was at 7.97 ppm, while for 9 the value reached
7.12 ppm. The chemical shifts of the proton for piperidinium
Surfactant
Salt R1
R2
R3
Statea contentb
1
2
3
4
5
6
7
8
9
CH3
CH2C6H5
CH2CH2OH CH2CH2OH
CH2CH2OH CH2CH2OH
C12H25/C14H29 Solid 98
cococ
Wax
Wax
99
99
Oleyld
(CH2CH2O)x (CH2CH2O)y
H-tallow f
Oleyld
Liquid 98
e
e
CH3
CH3
CH3
CH3
CH3
CH3
Wax
Wax
97
97
CH3
cococ
cococ
cococ
Liquid 98
Wax 99
Liquid 99
H-tallow f
C10H21
H-tallow f
C10H21
10 CH3
CH2QCHCH2 CH2C6H5
Liquid
Wax
—
99
11 C10H21
12 C12H25
13 C10H21
14 C12H25
—
—
—
—
—
—
—
—
Liquid 99
Liquid 99
Wax
99
a
b
c
At 25 1C. In %. Cocoalkyl chain distribution C8 – 5%, C10 – 6%,
d
C12 – 50%, C14 – 19%, C16 – 10%, and C18 – 10%. Oleyl chain
e
distribution C12 – 1%, C14 – 4%, C16 – 12%, and C18 – 82%. x + y = 15. theophyllinates (13–14) were lower, reaching 7.31 and 7.32 ppm
f
Alkyl hydrogenated tallow chain distribution C12 – 1%, C14 – 4%,
accordingly.
C16 – 31%, and C18 – 64%.
Data presented in Table 2 show that the prepared salts were
thermally stable. They manifested glass transition temperatures
in the range from À6 to À48 1C. Salts 1, 6, and 8 manifested no
glass transition temperature. In the case of 1, 6 and 8 the
crystallization and melting temperatures were observed. Decom-
position temperatures for all tested salts were very diverse and
changed in the range of 139–250 1C for Tonset 5% and 224–390 1C
for Tonset 50%, respectively.
The obtained room temperature ionic liquids (RTILs) were
subjected to the determination of density, viscosity and refrac-
tive index values in the temperature range of 20–80 1C. The
highest density value reached 1.2119 g cmÀ3 for 10, whereas the
lowest value reached 0.9448 g cmÀ3 for 7 (Table 3).
The viscosity values of RTILs at 20 1C varied notably depend-
ing on the structure of the cation (Table 4). The highest value
of 87.635 Pa s was observed for 10, while the lowest value of
0.370 Pa s was observed for 4. The differences in viscosity values
Fig. 1 Structure of prepared theophyllinates.
Table 3 Densitya of obtained RTILs
Table 2 Thermal transitions and decomposition temperaturesa of synthe-
sized theophyllinates
RTIL
d20
d40
d60
d80
b
c
d
e
e
Salt
Tg
Tc
Tm
Tonset 5%
Tonset 50%
4
7
9
10
12
13
1.0945
0.9448b
0.9699
1.2119
1.0575
1.0973
1.0805
0.9354
0.9575
1.1987
1.0447
1.0841
1.0662
0.9228
0.9449
1.1852
1.0316
1.0709
1.0517
0.9101
0.9320
1.1710
1.0181
1.0577
1
3
4
5
6
7
8
9
—
76
—
—
—
116
—
161
250
208
196
206
195
248
184
139
269
357
390
288
236
253
392
224
288
À6
À48
À28
—
—
—
À10, 5
À0, 7
a
b
In g cmÀ3, at 20, 40, 60 and 80 1C. At 25 1C.
À35
—
—
22
—
—
—
22
À46
À10
—
—
10
Table 4 Viscositya of obtained RTILs
a
b
c
In 1C. Glass transition temperature. Crystallization temperature.
Melting point upon heating. Decomposition temperatures as Tonset 5% to
d
e
RTIL
Z20
Z40
Z60
Z80
5% and Tonset 50% to 50% mass loss.
4
7
9
10
12
13
0.370
0.078
0.218
0.164
4.126
0.303
0.611
0.026
0.054
0.042
0.407
0.073
0.104
0.012
0.020
0.017
0.074
0.026
0.035
0.755b
1.089
popular organic solvents. They were hygroscopic and require
drying under reduced pressure at elevated temperature. The ILs
could be made anhydrous by heating at 80 1C in a vacuum
and storing them over P4O10. Upon such treatment of salts,
87.635
2.587
14.157
a
b
Pa s, at 20, 40, 60 and 80 1C. At 25 1C.
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Table 6 Criteria for the estimation of feeding deterrent activity based on
the total coefficient
Total coefficient
Deterrent activity
200–151
150–101
100–51
50–0
Very good
Good
Medium
Week
Fig. 2 Refractive index of didecyldimethylammonium theophyllinate (9).
Table 7 Feeding deterrent activities and the total coefficient of prepared
theophyllinates
Tribolium
confusum
(larvae)
Trogoderma
granarium
(larvae)
Tribolium
confusum
(weevils)
Sitophilus
granarius
(adults)
diminished with increasing temperature and reached a similar
level ranging from 0.074 to 0.012 Pa s at 80 1C. The values of the
refractive index for the obtained RTILs varied in the range of
1.5290–1.4760. The dependence of the refractive index on
temperature was linear – with the increase of temperature the
refractive index values decreased, which can be seen in Fig. 2
for 9.
Compound
1
2
3
4
7
9
11
12
163.3a
169.3a
178.2a
146.7b
158.2a
187.8a
155.3a
155.3a
131.3b
161.6a
73.2c
181.8a
160.7a
186.0a
178.3a
194.7a
191.3a
190.6a
186.8a
187.4a
174.6a
149.1b
145.3b
180.7a
130.4b
153.6a
167.2a
168.6a
143.8b
106.7b
159.7a
88.7c
166.0a
141.3b
168.9a
169.3a
142.8b
147.4b
139.8b
141.4b
150.0b
150.0b
66.1c
Table 5 presents the parameters of surface activity: critical
micelle concentration CMC, surface tension at CMC gCMC
surface excess concentrations at the saturated interface Gmax
,
,
13
14
121.2b
87.9c
Theophylline
Potassium
theophyllinate
Azadirachtin
the minimum surface occupied by a molecule at the interface
Amin and the Gibbs free energy of the adsorption layer DGa0ds
62.9c
106.4b
65.8c
.
188.4a
194.2a
185.0a
174.3a
It was found that the type of cation was decisive for CMC of
aqueous solutions of the synthesized salts. In the case of the
same anion, the cation exchange caused a decrease of CMC
values, e.g. from 38.9 and 11.8 mmol dmÀ3 for piperidinium
(13–14) to 6.92 and 0.34 mmol dmÀ3 for ammonium theo-
phyllinates. It was observed that elongation of alkyl substituents
in the cation contributed to the decrease of CMC values. In
ammonium theophyllinate water solutions (1–7, 9) the process of
micellization began at lower concentrations than in piperidinium
IL solutions. This was due to the presence of alkyl chain sub-
stituents longer than 8 carbon atoms in the cation of ammonium
theophyllinates (1–7, 9). The surface tension gCMC was reduced to
a minimum for theophyllinate (9), reaching 29.59 mN mÀ1. The
negative values of DG0ads for all studied theophyllinates indicate
that the process proceeds spontaneously.
a
b
c
Very good. Good. Medium.
the basis of the amount of food consumed. The activities were
estimated by the criteria listed in Table 6. The results are
presented in Table 7. All of the studied theophyllinates exhi-
bited insect-feeding deterrent activities (from very good to
medium, predominantly very good ones). Their activity was
higher compared to theophylline or potassium theophyllinate.
Compared to the standard (azadirachtin), their efficiency was
lower, with the exception of 3. By selecting the most appropriate
cation in combination with the theophyllinate as an anion, a
novel and effective insect-feeding deterrent may be obtained.
The obtained results are at a similar level of activity to those
obtained for the previously described ILs based on stored
product insect antifeedants, which consist of tripolyphoshate,
dihydrogen citrate and propionate anions.23
Insect-feeding deterrent activities of synthesized theophyllinates
towards Tribolium confusum (larvae and adults), Sitophilus granarius
(adults), and Trogoderma granarium (larvae) were presented on
Antifungal activity of the tested ILs was evaluated in respect of
four species of pathogenic fungi: Fusarium culmorum, Sclerotinia
sclerotiorum, Microdochium nivale and Botrytis cinerea. In the
preliminary experiment with 9 the mycelial growth of the tested
fungi was significantly inhibited at concentrations of 10, 100
and 1000 ppm. All the tested ILs (except 8) showed fungistatic
activity at a concentration of 1000 ppm, which can be seen
in Table 8.
A significant part of the salts also inhibited the growth of
mycelia at a concentration of 100 ppm. The fungus of M. nivale
was most sensitive to the applied ILs at a concentration of
10 ppm. The tested theophyllinates, particularly at a concentration
of 10 and 100 ppm, were less effective in comparison to the
commercial fungicide Tebu 250 EW containing tebuconazole.
At a concentration of 1000 ppm their efficiency was most often
Table 5 Surface activity of prepared theophyllinates
CMCa
gCMC
Gmax
Amin
ÀDG0ads
b
c
d
e
Salt
1
2
3
4
5
6
7
9
11
12
13
14
2.69
2.10
0.79
0.34
0.65
1.99
0.37
1.23
6.92
5.62
38.9
11.8
35.38
34.52
38.86
49.61
41.80
33.19
30.07
29.59
36.57
37.81
32.00
39.86
3.27
3.60
1.93
2.44
4.05
4.39
3.57
4.33
7.90
7.02
5.07
4.61
8.61
6.81
4.10
3.78
4.65
3.84
2.10
2.36
1.24
1.44
26.02
25.48
34.27
28.79
24.57
21.65
30.21
26.20
16.28
17.95
10.86
13.66
13.4
11.5
a
b
c
d
e
mmol dmÀ3
.
mN mÀ1
.
106 mol mÀ2
.
1019 m2. kJ mÀ1
.
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Table 8 Inhibition of the growth of Fusarium culmorum, Microdochium nivale, Botrytis cinerea and Sclerotinia sclerotiorum by prepared ILs
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The growth of F. culmorum
mycelium (cm)
The growth of M. nivale
mycelium (cm)
The growth of B. cinerea
mycelium (cm)
The growth of S. sclerotiorum
mycelium (cm)
IL
10 ppm 100 ppm 1000 ppm 10 ppm 100 ppm 1000 ppm 10 ppm 100 ppm 1000 ppm 10 ppm 100 ppm 1000 ppm
Control
2
3
4
5
6
7
8
11
12
13
14
4.6
4.4
4.6
4.6
4.2
4.2
4.6
4.6
4.6
4.6
4.6
4.6
4.6
2.7
2.8
3.0
4.3
2.1
4.6
4.6
4.6
3.8
4.6
4.6
0.0
0.59
4.6
1.8
2.3
2.2
2.2
0.0
2.6
4.6
0.0
0.9
0.2
0.0
0.0
0.33
4.6
4.2
1.9
3.2
3.1
4.0
4.6
4.6
4.0
1.7
4.6
3.5
1.2
0.28
4.6
2.0
1.4
0.7
1.7
2.1
3.3
4.6
1.6
0.6
2.9
1.8
0.0
0.35
4.6
0.4
0.4
0.2
0.3
0.1
1.2
3.6
0.1
0.0
0.5
0.0
0.0
0.21
4.6
4.6
4.6
4.6
4.6
4.4
4.6
4.6
4.6
4.6
4.6
4.6
3.4
0.08
4.6
4.6
4.6
2.5
4.6
4.4
4.6
4.6
3.0
1.8
4.6
3.7
1.0
0.33
4.6
0.1
1.2
0.0
1.2
0.0
1.7
4.6
0.0
0.0
0.0
0.0
0.0
0.20
4.6
4.6
4.6
4.6
4.6
4.6
4.2
4.6
4.6
4.6
4.6
4.6
0.3
0.28
4.6
1.2
2.3
2.4
2.4
0.6
4.6
4.6
2.0
0.6
4.6
0.3
0.0
0.37
4.6
0.1
0.2
0.1
0.3
0.0
0.8
4.6
0.0
0.0
0.0
0.0
0.0
0.16
Tebu 250 EW 0.1
LSD(P=0.05) 0.18
very high. This suggests that the obtained salts are novel and decyl, 2-hydroxyethyl, polyoxyethylene and allyl. The number
efficient antifungal ILs. The selection of the ammonium cation, and length of substituents in the cation should be taken into
which exhibits notable antibacterial and antifungal activities consideration during the design of biodegradable ammonium
even as a chloride, was relevant and ensured high activity against theophyllinates. Some reports suggest that Pseudomonas putida
fungi. The high activity of ILs with an anion originating from is capable of utilizing these compounds as sole carbon sources,24
natural resources towards fungi leads to a question regarding however this was not observed in the framework of this study.
their biodegradability.
The foundation of this phenomenon may be related to the
The determination of residues after biodegradation tests fact that theophyllinates exhibit higher resistance compared to
revealed that the theophylline anion was not biodegraded in all theophylline.
of the salts. Furthermore, the biodegradation of the cation for
The employed PCR method was used to evaluate whether the
theophyllinates 1–6, 9 and 10 was also marginal. ILs 7 and 8 presence of the studied salts contributed to the facilitation or
were characterized by low polarity and hence it was not possible inhibition of microbial growth. The results are shown in Fig. 3.
to determine their residues using the employed method. The
The PCR assay indicated that the studied theophyllinates
progress of biodegradation processes was only observed for had a negative impact on the bacterial species in the consortium,
the cation of a single IL (2). The cocoalkyl substituent of this since a strong inhibition of microbial growth could be observed
cation is a mixture of linear, saturated alkyls with various chain in general. It is worth noting that some of the salts exhibit
lengths from 8 to 18 carbon atoms and unsaturated alkyls with selective inhibitory activity against certain bacterial species – i.e.
18 carbon atoms. The biodegradation efficiency for the cation ILs 3 and 4 were most efficient in decreasing the growth of
of 2 is shown in Table 9. The highest susceptibility to bio- Variovorax species (VariP), 8 was effective against Sphingobacterium
degradation was observed for the longest alkyl chain (C18), (SphiP), while 10 notably limited the growth of Achromobacter
whereas the shortest alkyl chain (C8) was characterized by the species (AchrP). Facilitation of microbial growth occurred
lowest biodegradability. The biodegradation experiments carried exclusively for theophyllinate 2. In this IL, the highest increase
out with the ammonium theophyllinates (1–10) highlight the in the relative abundance was observed for Variovorax (VariP),
crucial role of substituents at the quaternary nitrogen atom. The
studied salts possessed different substituents, such as: methyl,
benzyl, a mixture of saturated and unsaturated straight-chain
alkyls (coco, oleyl, hydrogenated tallow and C12H25/C14H29),
Table 9 Biodegradation efficiency of linear alkyl substituents with various
chain lengths in the cation of theophyllinate (2)
Cocoalkyl chain substituent
Chain length
Contenta
Biodegradation efficiencya
C8
5
6
50
19
10
10
13.7
16.0
16.9
33.8
66.9
73.1
C10
C12
C14
C16
C18
Fig. 3 Analysis of qualitative and quantitative changes in the relative abun-
dance of species in the bacterial consortium after biodegradation tests with
the selected theophyllinates (1–10).
a
In %.
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while the lowest increase was observed for Sphingobacterium 20 cm3 of anhydrous acetone. KCl or KBr (residue of the
(SphiP). exchange reaction) was separated by filtration and acetone
The results obtained with the use of real time PCR were in was evaporated to obtain the product, which was finally dried
accordance with the determination of biodegradation residues under reduced pressure at 50 1C.
via HPLC-MS. The majority of the studied theophyllinates were
characterized by a marginal susceptibility to biological degra- [D6]DMSO, 20 1C, TMS): d = 0.85 (m, 3H; CH3), 1.24 (s, 18H;
Benzalkonium theophyllinate (1). 1H NMR (300 MHz,
dation and exhibited a negative impact on microbial growth.
CH2), 1.77 (s, 2H; CH2), 3.01 (s, 6H; CH3), 3.21 (s, 3H; CH3), 3.41
(s, 3H; CH3), 4.64 (s, 2H; CH2), 7.25 (s, 1H; CH), 7.51 (t, 3J(H,H) =
12.4 Hz, 2H; CH), 7.60 (m, 3H; CH); 13C NMR (75 MHz,
[D6]DMSO, 20 1C, TMS): d = 13.9, 21.8, 22.1, 25.8, 27.3, 28.6,
31.3, 48.9, 63.3, 65.9, 114.3, 128.2, 128.7, 130.1, 132.9, 146.0,
149.6, 151.7, 157.2.
Experimental
Materials
Theophylline, didecyldimethylammonium chloride, and benz-
alkonium chloride were obtained from Sigma Aldrich/Fluka
and used without further purification. Quaternary ammonium
chlorides with oleyl, hydrogenated tallow and coco alkyl groups
are products of AkzoNobel. Allylbenzyldimethylammonium bromide,
1-alkyl-1-methylpiperidinium bromide and alkylcyclohexyldimethyl
ammonium bromide were synthesized according to the procedure
described recently.25
Cocodi(2-hydroxyethyl)methylammonium theophyllinate (2).
1H NMR (300 MHz, [D6]DMSO, 20 1C, TMS): d = 0.86 (t, 3J(H,H) =
6.6 Hz, 3H; CH3), 1.25 (m, 20H; CH2), 1.65 (s, 2H; CH2), 3.19
(s, 6H, CH3), 3.37 (t, 3J(H,H) = 7.9 Hz, 5H; CH3, OH), 3.58
(t, 3J(H,H) = 3.9 Hz, 4H; CH2), 3.99 (t, J = 4.5 Hz, 4H; CH2),
7.21 (s, 1H; CH); 13C NMR (75 MHz, [D6]DMSO, 20 1C, TMS):
d = 13.9, 21.9, 22.3, 22.6, 26.3, 29.2, 29.3, 29.5, 29.6, 31.8, 48.8,
54.6, 63.6, 64.1, 111.6, 146.2, 149.7, 151.7, 157.2.
Oleyldi(2-hydroxyethyl)methylammonium theophyllinate (3).
1H NMR (300 MHz, [D6]DMSO, 20 1C, TMS): d = 0.86 (t, 3J(H,H) =
11.8 Hz, 3H; CH3), 1.24 (m, 22H; CH2), 1.67 (s, 2H; CH2), 1.98 (m,
3H; CH3), 3.07 (s, 3H; CH3), 3.21 (s, 3H; CH3), 3.38 (m, 2H; CH2),
3.43 (m, 11H; CH3, CH2, OH), 3.82 (d, 3J(H,H) = 4.9 Hz, 2H; CH2),
5.27 (s, 2H; CH), 7.27 (s, 1H; CH); 13C NMR (75 MHz, [D6]DMSO,
20 1C, TMS): d = 13.9, 21.7, 22.1, 25.8, 26.8, 27.6, 28.7, 28.8,
29.1, 29.7, 31.3, 48.9, 54.8, 63.2, 114.5, 129.6, 146.1, 149.7,
151.7, 157.3.
General
1H NMR spectra were recorded on a Mercury Gemini 300
spectrometer operating at 300 MHz with TMS as the internal
standard. 13C NMR spectra were obtained using the same instru-
ment at 75 MHz. CHN elemental analyses were performed at the
Adam Mickiewicz University, Poznan (Poland). The stability of the
synthesized salts in contact with air and water as well as their
solubility in various solvents was tested (detailed description given
in the ESI†). The water content was determined using an Aquastar
volumetric Karl-Fischer titrator with composite 5 solution as
the titrant and anhydrous methanol as a solvent. Density was
measured using an Automatic Density Meter DDM2911 using the
mechanical oscillator method. Density of the samples (approx.
2.0 cm3) was measured with respect to temperature controlled via
Peltier, from 20 to 90 1C. The uncertainty of measurements was
estimated to be less than 10À5 g cmÀ3. Viscosity was determined
using a rheometer (Rheotec RC30-CPS) with cone-shaped geo-
metry (C50-2). The viscosity of the samples (approx. 1.5 cm3) was
measured with respect to temperature, from 20 to 90 1C. The
uncertainty of the viscosity measurement was estimated to be less
than 10À4 Pa s. The refractive index was determined using an
automatic refractometer J357 with electronic temperature control
from 20 to 90 1C. The uncertainty of measurements was estimated
Polyoxyethylene (15) (hydrogenated tallow)methylammonium
theophyllinate (4). 1H NMR (300 MHz, [D6]DMSO, 20 1C, TMS):
d = 0.86 (t, 3J(H,H) = 6.7 Hz, 3H), 1.24 (m, 30H; CH2), 1.63 (s, 2H;
CH2), 3.07 (s, 3H; CH3), 3.22 (s, 3H; CH3), 3.43 (m, 5H; CH3,
OH), 3.51 (m, 2H; CH2), 3.55–3.6 (m, 52H; CH2), 3.82 (s, 3J(H,H) =
4.6 Hz, 4H; CH2), 4.00 (s, 4H; CH2), 7.26 (s, 1H; CH); 13C NMR
(75 MHz, [D6]DMSO, 20 1C, TMS): d = 13.9, 21.7, 22.1, 28.7, 28.8,
29.1, 31.3, 48.8, 60.2, 62.7, 69.5, 69.4, 69.8, 72.4, 114.4, 146.2,
149.6, 151.8, 157.5.
Oleyltrimethylammonium theophyllinate (5). 1H NMR
3
(300 MHz, [D6]DMSO, 20 1C, TMS): d = 0.86 (t, J(H,H) = 11.8 Hz,
3H, CH3), 1.24 (m, 22H; CH2), 1.67 (s, 2H; CH2), 2.01 (m, 3H; CH3),
3.07 (s, 9H; CH3), 3.21 (s, 3H; CH3), 3.43 (m, 3H; CH3), 3.82
(d, 3J(H,H) = 4.9 Hz, 2H; CH2), 5.27 (s, 2H; CH), 7.27 (s, 1H; CH);
13C NMR (75 MHz, [D6]DMSO, 20 1C, TMS): d = 13.9, 21.7, 22.1,
25.8, 26.9, 27.6, 28.7, 28.8, 29.1, 29.7, 31.5, 48.6, 63.2, 114.5,
129.7, 146.3, 149.6, 151.5, 157.1.
to be less than 10À5
.
Cocotrimethylammonium theophyllinate (6). 1H NMR
Synthesis
3
Quaternary ammonium chlorides or bromides or 1-alkyl-1- (300 MHz, [D6]DMSO, 20 1C, TMS): d = 0.86 (t, J(H,H) = 6.7 Hz,
methylpiperidinium bromides (0.05 mol) were dissolved in 3H; CH3), 1.25 (m, 20H; CH2), 1.60 (m, 2H; CH2), 3.21 (s, 3H; CH3),
methanol and a stoichiometric amount of saturated methanol 3.27 (s, 9H; CH3), 3.30 (t, 3J(H,H) = 8.5 Hz, 2H; CH2), 3.43 (m, 3H;
solution of KOH was added. The solutions were stirred at room CH3), 7.24 (s, 1H; CH); 13C NMR (75 MHz, [D6]DMSO, 20 1C, TMS):
temperature for 5 min, after which the precipitated KCl or KBr d = 13.9, 22.7, 23.1, 26.2, 29.25, 29.32, 29.4, 29.5, 29.6, 31.9,
was filtered off. Then, a stoichiometric amount of an appro- 49.8, 62.5, 114.7, 146.6, 149.4, 151.7, 157.3.
priate theophylline was added. The solutions were stirred again
at room temperature for 15 min and after evaporation of the (300 MHz, [D6]DMSO, 20 1C, TMS): d = 0.86 (t, J(H,H) = 8.7 Hz,
solvent at a temperature of 40 1C, the product was dissolved in 6H; CH3), 1.25 (s, 44H; CH2), 1.61 (m, 4H; CH2), 3.02 (s, 6H; CH3),
Dicocotrimethylammonium theophyllinate (7). 1H NMR
3
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3.19 (s, 7H; CH2, CH3), 3.38 (s, 6H; CH3), 7.16 (s, 1H; CH); 3.28–3.30 (m, 6H; CH2), 3.41 (s, 3H; CH3), 7.32 (s, 1H; CH); 13
C
13C NMR (75 MHz, [D6]DMSO, 20 1C, TMS): d = 13.9, 21.7, 22.1, NMR (75 MHz, [D6]DMSO, 20 1C, TMS): d = 13.90, 19.28, 20.68,
25.7, 27.2, 28.5, 28.7, 28.9, 29.7, 31.3, 49.9, 62.7, 114.8, 146.3, 20.96, 22.10, 25.87, 27.33, 28.56, 28.74, 28.86, 28.96, 29.03,
149.7, 151.8, 157.4.
29.04, 29.76, 31.31, 46.81, 59.84, 62.44, 113.48, 145.41, 149.37,
Di(hydrogenated tallow alkyl)dimethylammonium theophyl- 151.68, 156.93; elemental analysis calcd (%) for C25H45N5O2: C
linate (8). 1H NMR (300 MHz, [D6]DMSO, 20 1C, TMS): d = 0.87 67.08, H 10.13, N 15.64; found: C 67.29, H 10.27, N 15.96.
(t, 3J(H,H) = 6.7 Hz, 6H; CH3), 1.26 (m, 48H; CH2), 1.61 (m, 4H; CH2),
3
Thermal analysis
3.23 (s, 3H; CH3), 3.31 (s, 6H; CH3), 3.39 (t, J(H,H) = 6.3 Hz, 7H;
CH2, CH3), 7.19 (s, 1H; CH). 13C NMR (75 MHz, [D6]DMSO, 20 1C, Thermal transition temperatures of the obtained salts were
TMS): d = 14.0, 22.5, 26.1, 27.2, 29.1, 29.25, 29.29, 29.37, 29.50, 29.55, determined by DSC, using a Mettler Toledo Stare TGA/DSC1
29.59, 31.8, 51.03, 63.2, 114.8, 146.3, 149.7, 151.8, 157.4.
(Leicester, UK). Samples between 5 and 15 mg were placed in
Allylbenzyldimethylammonium theophyllinate (10). 1H NMR aluminum pans and heated from 25 to 120 1C at a heating rate
(300 MHz, [D6]DMSO, 20 1C, TMS): d = 2.92 (s, 6H; CH3), 3.22 (s, of 10 1C minÀ1 and cooled using an intracooler at a cooling rate
3
3H; CH3), 3.41 (s, 3H; CH3), 3.97 (d, J(H,H) = 7.3 Hz, 2H; CH2), of 10 1C minÀ1 to À100 1C. Thermogravimetric analysis was
4.52 (m, 2H; CH2), 6.15 (m, 1H; CH), 7.22 (s, 1H; CH), 7.57 (m, 5H; performed using a Mettler Toledo Stare TGA/DSC1 unit (Leicester,
CH); 13C NMR (75 MHz, [D6]DMSO, 20 1C, TMS): d = 27.4, 29.5, UK) under nitrogen. Samples between 2 and 10 mg were placed
48.6, 65.8, 66.5, 125.7, 127.9, 128.7, 130.2, 132.9, 114.2, 146.2, in aluminum pans and heated from 30 to 450 1C at a heating rate
149.5, 151.6, 157.3; elemental analysis calcd (%) for C19H25N5O2 C of 10 1C minÀ1. Nitrogen was used as a carrier gas.
64.20, H 7.09, N 19.70; found: C 64.41, H 7.28, N 19.41.
Surface activity
Cyclohexyldecyldimethylammonium theophyllinate (11).
1H NMR (300 MHz, [D6]DMSO, 20 1C, TMS): d = 0.89 (t, The surface tension was determined using the shape drop method.
3J(H,H) = 6.4 Hz, 3H; CH3), 1.12 (m, 1H; CH), 1.39–1.49 (m, The measurements were carried out by the use of a DSA 100
20H; CH2), 1.69–1.74 (m, 1H; CH), 2.02 (m, 2H; CH2), 2.19 (m, analyzer (Kru¨ss, Germany, the accuracy of 0.01 mN mÀ1) at 25 1C
2H; CH2), 3.30 (s, 6H; CH3), 3.48 (t, 3J(H,H) = 6.2 Hz, 2H; CH2), (temperature controlled using a Fisherbrand FBH604 thermostatic
3.50–3.53 (m, 1H; CH), 3.40 (s, 6H; CH3), 7.28 (s, 1H; CH). bath – Fisher, Germany, with the accuracy of 0.1 1C). The principle
13C NMR (75 MHz, [D6]DMSO, 20 1C, TMS): d = 13.2, 19.2, 24.0, of this method is to form an axisymmetric drop at a tip of a syringe
24.1, 24.7, 25.8, 28.7, 28.8, 28.9, 29.1, 30.6, 48.2, 62.0, 71.5. needle. The image of the drop (3 cm3) acquired using a CCD
114.22, 145.91, 149.56, 151.74, 157.17; elemental analysis calcd camera was digitized. The surface tension (gCMC, mN mÀ1) was
(%) for C25H45N5O2: C 67.08, H 10.13, N 15.64; found: C 67.30, calculated by analyzing the profile of the drop according to
H 10.27, N 15.36.
Laplace’s equation. The values of the critical micelle concen-
Cyclohexyldodecyldimethylammonium theophyllinate (12). tration (CMC) and the surface tension at the CMC (gCMC) were
1H NMR (300 MHz, [D6]DMSO, 20 1C, TMS): d = 0.86 (t, 3J(H,H) = determined from the intersection of the two straight lines
6.0 Hz, 3H; CH3), 1.07–1.14 (m, 1H; CH), 1.24–1.32 (m, 20H; drawn in low and high concentration regions in surface tension
CH2), 1.40–1.46 (m, 2H; CH2), 1.57–1.66 (m, 3H; CH, CH2), curves (gCMC vs. log C curves) using a linear regression analysis
1.81–1.85 (m, 2H; CH2), 2.03–2.07 (m, 2H; CH2), 2.96 (s, 6H; method.
CH3), 3.24 (m, 1H; CH), 3.27–3.31 (m, 2H; CH2), 3.40 (s, 6H;
CH3), 7.22 (s, 1H; CH); 13C NMR (75 MHz, [D6]DMSO, 20 1C,
Assay of biological activity as potential fungicides
TMS): d = 13.93, 21.54, 22.11, 24.38, 24.83, 25.29, 25.84, 27.3, Four species of fungi were used: Fusarium culmorum (KZF-5),
28.52. 28.73, 28.87, 28.95, 29.03, 29.04, 29.77, 31.31, 47.60, Sclerotinia sclerotiorum (KZF-14), Microdochium nivale (KZF-7)
61.67, 70.58, 114.22, 145.91, 149.56, 151.74, 157.17; elemental and Botrytis cinerea (KZF-12) (obtained from the Institute of
analysis calcd (%) for C27H49N5O2: C 68.17, H 10.38, N 14.72; Plant Protection-NRI collection).
found: C 68.01, H 10.62, N 14.51.
The sample of tested salts was dissolved in 4 cm3 of water,
1
1-Decyl-1-methylpiperidinium theophyllinate (13). H NMR then added to sterile medium (PDA – Potato Dextrose Agar,
(300 MHz, [D6]DMSO, 20 1C, TMS): d = 0.86 (t, 3J(H,H) = 5.2 Hz, Difcot) and cooled to 50 1C. The salt concentration in the
3H; CH3), 1.23 (m, 14H; CH2), 1.51–1.56 (m, 2H; CH2), 1.61–1.64 medium was 10, 100 and 1000 ppm. Liquid medium containing
(m, 2H; CH2), 1.77 (m, 4H; CH2), 3.01 (s, 3H; CH3), 3.22 (s, 3H; the tested salts was distributed on the Petri dishes of diameter
CH3), 3.27–3.31 (m, 6H; CH2), 3.42 (s, 3H; CH3), 7.31 (s, 1H; CH); 50 mm. The 4 mm disks of the examined fungi were placed in
13C NMR (75 MHz, [D6]DMSO, 20 1C, TMS): d = 13.89, 19.27, 20.65, the center of the Petri dish. In the control sample the fungi
20.91, 22.15, 25.83, 27.27, 28.51, 28.69, 28.82, 28.96, 29.04, 29.68, were grown on PDA with the addition of sterile water. The
31.26, 46.85, 59.79, 62.44, 113.42, 145.45, 149.38, 151.68, 156.91; tested compounds were compared with a commercial fungicide
elemental analysis calcd (%) for C23H41N5O2: C 65.83, H 9.85, (Tebu 250 EW) containing tebuconazole as an active substance.
N 16.69; found: C 65.57, H 9.65, N 17.01.
1-Dodecyl-1-methylpiperidinium theophyllinate (14). H NMR in the control reached the edge of the Petri dish. Afterwards, the
(300 MHz, [D6]DMSO, 20 1C, TMS): d = 0.86 (t, J = 5.9 Hz, 3H; diameter of the mycelium was measured, subtracting the initial
The plates were incubated at room temperature until the mycelium
1
3
CH3), 1.24 (m, 18H; CH2), 1.51–1.56 (m, 2H; CH2), 1.61–1.65 (m, diameter of the disc with the fungus (4 mm). For each experimental
2H; CH2), 1.78 (m, 4H; CH2), 3.01 (s, 3H; CH3), 3.21 (s, 3H; CH3), sample four replications were performed. The results were subjected
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to Student–Newman–Keuls’s analysis to test for significant differ- weighed and offered to the insects in plastic boxes as the sole food
ences between control and samples with addition of ILs.
source for 5 days. In all variants, three deterrent coefficients were
calculated as follows: absolute coefficient of deterrency, A =
(CC À TT)/(CC + TT) Â 100; relative coefficient of deterrency, R =
Microorganisms
A bacterial consortium with a high biodegradation potential (C Â T)/(C + T) Â 100; and the total coefficient of deterrency, the sum
towards studied salts was selected from the group of 218 consortia of the absolute and relative coefficients. In these equations, CC is the
isolated from petroleum-contaminated soil.26 The genetic charac- average weight of the food consumed in the control, TT is the
terization of the bacterial consortium based on the analysis of 16s average weight of the food consumed in the no-choice test and T and
rRNA sequences revealed the following taxa: Alcaligenes (AlcP); C are the average weights of the food consumed in the choice test.
Sphingobacterium (SphiP); Citrobacter (CKK); Achromobacter Each of the three experiments was repeated five times with 3 adults
(AchrP); Comamonadaceae (ComP); Pseudomonas (PseuP); and of Sitophilus granarius, 20 adults and 10 larvae of Tribolium
Variovorax (VariP). Preparation of the inoculum and subsequent confusum, and 10 larvae of Trogoderma granarium. The number of
cultivation conditions were the same as described recently27 individual insects depended on the intensity of their food consump-
(detailed description given in the ESI†).
tion. The adults used for the experiments were unsexed, 7–10 days
old, and the larvae were 15–30 days old. After 5 days the discs were
reweighed and the average weight of eaten food was calculated.
Preparation of biodegradation tests
The biodegradation tests were carried out in loosely closed
Erlenmeyer flasks, with 50 cm3 of the mineral medium and
approx. 0.025 g of the studied salt, which is a sole source of carbon
and energy. The initial inoculum was adjusted to reach an OD600
value of 0.1 Æ 0.01 by adding approx. 1 cm3 of dense cell
suspension from the aerobically grown preculture. The cultivation
was carried out at 25 1C and 120 rpm for 30 days. The samples
were prepared in triplicates. Samples lacking biomass served as
control to account for potential abiotic losses. After finishing the
biodegradation tests, the biomass was separated by centrifugation
(10 000g for 10 min) and was rinsed three times with the mineral
medium (5 cm3). The aliquots were combined with the super-
natant. About 10 cm3 of the supernatant were subjected to
ultrasound-assisted extraction with methanol (3 Â 1 cm3). The
extracts were combined, filtered through a 0.2 mm PTFE syringe
filter, diluted with methanol : water solution (80 : 20 v/v) and sub-
jected to determination of residual salts with the use of HPLC-MS
(UltiMate 3000 RSLC, Dionex, with a Hypersil GOLD column
100 mm  2.1 mm I.D.; 1.9 mm and an API 4000 QTRAP triple
quadrupole mass spectrometer, AB Sciex).
Conclusions
Third generation ionic liquids were synthesized by employing a
natural product, theophylline, as an anion. The physical state of
the synthesized theophyllinates depends on the size of the cation.
Salts with a melting point above 100 1C and ionic liquids in the
form of liquids and waxes may be obtained. They are stable in air
as well as in contact with water and popular organic solvents.
At the same time they are thermally stable and display surface
activity. All synthesized theophyllinates exhibited insect-feeding
deterrent activities (from very good to medium, predominantly
very good). Very efficient antifungal properties could be observed
at a concentration of 1000 ppm, hence the obtained salts are
novel fungicidal and potentially pesticidal ionic liquids. High
biological resistance of the tested theophyllinate-based ILs may
be advantageous in terms of applicability. They may be used in
specific biocidal agents and remain active even after prolonged
time periods. The obtained data also suggest that certain struc-
tural modifications may increase the biodegradability in order to
provide an environmentally friendly product. On the basis of IL 2,
it can be established that long, saturated and unsaturated alkyl
substituents in the cation enhance the biodegradability of theo-
phyllinate, which corresponds well with the observations of other
researchers.29 The synthesized theophyllinate-based ILs are sur-
face active compounds, however it is difficult to correlate the
surface activity with the established biological activities.
Analysis of qualitative and quantitative changes in the relative
abundance of species in the bacterial consortium with the use
of real time PCR
Bacterial DNA extracted from the biomass obtained after the
biodegradation tests was subjected to real-time PCR by the ddCt
method for relative quantification to study bacterial community
dynamics, as described recently.27
Feeding deterrent activity test
Acknowledgements
The bioassay experiments were conducted with Tribolium confusum
Duv. (larvae and adults), Sitophilus granarius L. (adults), and
Trogoderma granarium Ev. (larvae). The insects were grown on a
wheat grain or whole-wheat meal diet in laboratory colonies, which
were maintained at 26 Æ 1 1C and 60 Æ 5% relative humidity. Choice
and no-choice tests for insect-feeding were conducted following a
previously described procedure.28 Wheat wafer discs (1 cm diameter,
1 mm thick) were saturated by dipping either in ethanol only
(control) or in an ethanol solution of the studied salts (1%). After
evaporation of the solvent (30 min of air-drying) the wafers were
This work was supported by grant NCN No. DEC-2012/07/B/ST5/
00806 and No. DEC-2011/03/B/NZ9/00731.
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